Collagen/polysaccharide bilayer matrix

ABSTRACT

Disclosed are bilayer matrices of a polysaccharide such as collagen (COL) and another polysaccharide such as hyaluronic acid (HA) with various COL/HA ratios. Each layer has a porous structure. These materials are useful for tissue regeneration, particularly when used with orthopedic implants and drug delivery.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior application Ser. No.:09/652,604, entitled, “COLLAGEN/POLYSACCHARIDE BILAYER MATRIX” by EdwardRobert C. Spiro and Lin Shu Liu, filed on Aug. 30, 2000 now U.S. Pat.No. 6,773,723.

BACKGROUND OF THE INVENTION

The present invention is directed to biodegradable matrices for tissueregeneration.

Polysaccharides, such as glycosaminoglycans that include hyaluronic acid(HA) have been used in a wide variety of biomaterials. Hyaluronic acid(HA), a naturally-occurring polysaccharide, has been used in matrixengineering in ophthalmic and orthopedic medicine. Clinical indicationsfor HA alone are limited by its physical properties and the shortresidence time of the natural HA molecule. A formaldehyde cross-linkedHA, Hylan, has been used in viscosupplementation of arthritic diseasedjoints (Takigami et al., 1993, Carbohydrate Polymers 22: 153-160.

Berg et al., (U.S. Pat. No. 5,510,418, issued Apr. 4, 1996) discloseglycosaminoglycans, such as, HA, chondroitin sulfates, keratan sulfates,chitin and heparin, chemically conjugated to a synthetic hydrophilicpolymer, such as polyethylene glycol (PEG) that are used as injectableformulations or solid implants. Kimata et al., (U.S. Pat. No. 5,464,942issued Nov. 7, 1995) disclose phospholipid linked glycosaminoglycans andtheir use as metastasis inhibitors. Sakurai, et al, U.S. Pat. No.5,310,881 issued May 10, 1994, disclose glycosaminoglycan-modifiedproteins. Balazs et al., U.S. Pat. No. 5,128,326 issued Jul. 7, 1992,disclose hyaluronan cross-linked with divinyl sulfone.

SUMMARY OF THE INVENTION

The present invention provides biodegradable matrices for tissueregeneration, methods of making the matrices and methods of using thematrices.

A biodegradable matrix of the present invention comprises two layers,each layer comprising a cross-linked polymeric component that differ intheir composition, density, and porosity, wherein each of the polymericcomponents is a derivative of a member selected from the groupconsisting of collagen, albumin, fibrinogen, fibronectin, vitronectin,laminin, hyaluronic acid, dextran, dextran sulfate, chondroitin sulfate,dermatan sulfate, keratan sulfate, chitin, chitosan, heparin, heparinsulfate and alginate.

The two layers are attached by either mechanical adhesion or chemicalcross-linking.

The biodegradable matrices are made by forming a layer by reacting apolymeric component with a cross-linking agent such as divinyl sulfoneor a dialdehyde. Then the second layer, which may be a slurry, isapplied and may either mechanically adhere and gel onto the first layeror be chemically linked to the first layer by cross-linking agents. Thesecond layer also comprises a cross-linked polymeric component.

The present invention also provides a method of using the matrix toregenerate tissue by applying the matrix at a site of desired tissueregeneration.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE is a schematic flow chart of the synthesis of an embodimentof a bilayer matrix according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The matrices comprise at least two porous polymeric layers that differin their composition, density and porosity, so that they have differentcharacteristics within the environment of growing tissue. The twopolymeric layers are separately prepared, then assembled either bychemically cross-linking or mechanical embedding. The layers will differin at least one property among composition, density, porosity, and thenature of the cross-linking bond, but one or two of these properties maybe the same for both layers.

In the present invention each of the polymeric components is selectedfrom the group consisting of collagen, albumin, fibrinogen, fibronectin,vitronectin, laminin, hyaluronic acid, dextran, dextran sulfate,chondroitin sulfate, dermatan sulfate, keratan sulfate, chitin,chitosan, heparin, heparin sulfate and alginate. In a preferredembodiment, the polymeric component is a protein selected from the groupconsisting of collagen, albumin, fibrinogen, fibronectin, vitronectinand laminin. In another preferred embodiment the polymeric component isa polysaccharide selected from the group consisting of hyaluronic acid,dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratansulfate, heparin, heparin sulfate, chitosan, chitin and alginate. In apreferred embodiment, the polymeric components are selected from thegroup consisting of hyaluronic acid and collagen. As used herein, theterm “polymeric component” includes the polysaccharides or proteins andtheir salts such as the sodium, potassium, magnesium, calcium, and thelike, salts. Preferred forms of starting material of the polymericcomponents include those which have been approved for human use. Thestarting material for hyaluronate can be derived by bacterialfermentation or through isolation from rooster combs or can be obtainedfrom commercial sources.

Each layer may be comprised of the same or different polymericcomponents. In one preferred embodiment, the polymeric component in bothlayers is collagen. In another preferred embodiment, one layer comprisesHA and the other comprises collagen. Typically, the polysaccharides willhave an average molecular weight of about 1,000 to 10,000,000 DA.

A matrix of the present invention may be formulated in several physicalforms, including sponge-like forms.

Drugs, growth factors, polypeptides, proteins, cDNA, gene constructs andother bioactive therapeutic agents may also be included in the matrixand can be entrapped within the sponge either by mixing the agent withone of the two derivatives before gelatinization, or diffusion from adrug solution into the sponge after their formation.

The agent may also be covalently linked to the matrix.

The matrix may be formulated into a sponge-like material that isdesirable for an implantable formulation. The matrices of the presentinvention may be formed into any shape by lyophilization or air dryingin molds of the desired shape.

Growth factors and/or therapeutic agents may be included in the matrix,and can include proteins originating from various animals includinghumans, microorganisms and plants, as well as those produced by chemicalsynthesis and using genetic engineering techniques. Such agents include,but are not limited to, biologically active substances such as growthfactors such as, bFGF, aFGF, EGF (epidermal growth factor), PDGF(platelet-derived growth factor), IGF (insulin-like growth factor),TGF-β 1 through 3, including the TGF-β superfamily (BMP=s, GDF-5, ADMP-1and dpp); cytokines, such as various interferons, includinginterferon-alpha, -beta and -gamma, and interleukin-2 and -3; hormones,such as, insulin, growth hormone-releasing factor and calcitonin;non-peptide hormones; antibiotics; anti-cancer agents and chemicalagents, such as, chemical mimetics of growth factors or growth factorreceptors, and gene and DNA constructs, including cDNA constructs andgenomic constructs. In a preferred embodiment, the agents include thosefactors, proteinaceous or otherwise, which are found to play a role inthe induction or conduction of growth of bone, ligaments, cartilage orother tissues associated with bone or joints, such as for example, BMPand bFGF. The present invention also encompasses the use of autologousor allogeneic cells encapsulated within the matrix. The autologous cellsmay be those naturally occurring in the donor or cells that have beenrecombinantly modified to contain nucleic acid encoding desired proteinproducts.

As will be understood by those of skill in the art, the amount of agentto be immobilized or encapsulated within the carrier will vary dependingupon the intended target, but will usually be in the range of picogramto gram quantities.

A matrix of the present invention may be administered throughimplantation or direct application depending on the intendedapplication.

Each of the two polymeric layers may be respectively synthesized bycross-linking, for example, collagen and hyaluronic acid (COL and HA).Typical cross-linking agents include divinyl sulfone (DVS) andpolyaldehydes, such as, bi- or trialdehyde. If one of the layerscomprises a polysaccharide, it can be prepared for cross-linking byopening sugar rings and reacting with sodium periodate to produce apolysaccharide derivative with free aldehyde end groups. For chemicalassembling of two COL/HA polymeric layers with DVS, they should carryboth active hydrogen atoms and sulfone functional groups attached ontheir surfaces. This may be attained by either controlling the ratio ofCOL/HA to the cross-linker or varying the gelation time.

Alternatively, the layers may be linked by thermal dehydration. Formechanical attachment of the layers, one layer should have pores largeenough to allow the components of the second layer to penetrate wheregelation can take place. Thus, control of both the viscosity and theability to gel in solution of substances of the second layer areimportant. A slurry of the second layer material with low viscosity andlong gelation time may penetrate to the entire first layer. Theseparameters are controlled so the slurry penetrates sufficiently into thefirst layer to form a strong mechanical bond, where it gels.

In one embodiment, the first polymeric layer is prepared bycross-linking a polysaccharide or protein to another polysaccharide orprotein. The two polysaccharides or proteins may be the same ordifferent from one another. For example, collagen may be cross-linked tocollagen, or hyaluronate may be cross-linked to collagen. Various COL/HAratios may be used. Typical ratios are 2:8 to 9:1 collagen to HA.

The first polymeric layer may then be applied, mechanically and/orchemically, to the second polymeric layer. Typical chemical applicationmay be accomplished by cross-linking with DVS or a polyaldehyde linkingagent.

The FIGURE schematically shows an embodiment of the process for formingthe bilayer matrix. The collagen (COL) and hyaluronate (HA) areseparately cross-linked with DVS to form respectively the cross-linkedCOL layer 10 a and the cross-linked HA layer 10 b. The layer 10 a iscross-linked via hydroxy and amino groups on the peptide chains ofcollagen. The layer 10 b is cross-linked via hydroxy groups of thepolysaccharide. The layers 10 a and 10 b are then cross-linked to eachother with DVS to form the bilayer product 11.

The biologically active substance may be incorporated during fabricationof the matrix between cross-linking or mechanical application of layers.Alternatively, the biological substance may be incorporated after thematrix is fabricated by soaking the matrix in a solution containing theactive substance.

The efficacy of tissue regeneration can be shown by both in vitro and invivo tests known by those of ordinary skill in the art. In the presentinvention, the preferred therapeutic agents are those factors which arefound to play a role in the induction or conduction of growth of bone,ligaments, soft tissue, cartilage or other tissues associated with boneor joints. The matrix, which may include therapeutic agents as describedabove, will be applied at a site of desired tissue regeneration, such asbone growth, cartilage growth or joint repair.

In vitro and in vivo assays for the assessment of chondroinduction,chondroconduction, osteoinduction and osteoconduction are known by thoseof ordinary skill in the art. For the in vitro tests, primary fetal ratcalvarial cells, harvested by a series of collagenase digestions,according to the method of Wong and Cohn (PNAS USA 72:3167-3171, 1975),or primary rat epiphyseal cartilage, according to the method of Thybergand Moskalewski, (Cell Tissue Res. 204:77-94, 1979) or rabbit articularchondrocytes, harvested by the method of Blein-Sella O. et al., (MethodsMol. Biol., 43:169-175, 1995), are seeded into the carriers containingdesired agents and cultured under conventional conditions for 1-4 weeks.Cultures are then processed and evaluated histologically.

The chondroconductive or chondroinductive capability of a matrix. of thepresent invention can be determined by successful support of adhesion,migration, proliferation and differentiation of primary rat bone marrowand stromal cells as well as primary rat-or rabbit chondrocytes. Bonemarrow and bone marrow stromal cells are the source of chondroprogenitorcells found in the subchondral bone marrow of full-thickness defects.Bone marrow can be harvested from the long bones of 2-3 week-old inbredLewis rats and can be added directly to a carrier or cultured for 2weeks under standard conditions. The adherent stromal cell populationthat grows out of these cultures are passaged and frozen for use. Cellsfrom up to six passages are used for culturing or seeding on the carrierto test for chondroconductive or chondroinductive capabilities.

Retinoic acid-treated chondrocytes represent a less mature chondrocyteand can be used to test the ability of matrices to support later stagesof chondrogenesis. Retinoic acid treatment of primary chondocytes isperformed prior to culturing or seeding the cells on a carrer (bietz,.U. et al., 1993, J. Cell Biol. 52(1):57-68).

Cell adhesion and proliferation are monitored using an MTS assay thatcan measure cell number and viability based on mitochondrial activity.Stromal cells or chondrocytes are cultured on a carrier containing atherapeutic agent for 6-18 hrs. in the presence or absence of serum foradhesion analysis and for 1-2 weeks for proliferation assessment.

For cell migration testing, matrices are coated or fitted onto porousTrans-well membrane culture inserts (Corning). Stromal cells are seededon top of the carrier in the upper chamber of the Trans-well and achemoattractant (growth factor, PDGF) to placed in the bottom chamber.After 12-18 hrs of culture the cells that have migrated through thecarrier to the bottom side of the Trans-well membrane are quantitated bythe MTS assay. The matrix is removed from the upper chamber andprocessed histologically to assess the degree of infiltration.

The analysis of differentiation markers relevant to chondrogenesis andosteogenesis are evaluated at both the protein and transcriptionallevel. The specific markers that may be analyzed include: 1) Type IIcollagen and IIA, IIB isoforms; 2) Aggrecan proteoglycan; 3) Type is, Xand )G collagen; 4) Type I collagen; 5) Cartilage matrix protein (CMP);6) Cart-1 transcription factor; 7) Fibronectin (EDA, EDB isoforms); 8)Decorin proteoglycan; 9) Link protein; 10) NG-2 proteoglycan;. 11)Biglycan proteoglycan; 12) Alkaline phosphatase. Differentiation may bemeasured by Northern/PCR analysis, Western blotting or by metabolic celllabeling.

For Northern/PCR analysis, RNA is isolated by standard procedures fromstromal cells or chondrocytes. Time course tests may be used todetermine optimal culture periods that range from 1 to 6 weeks dependingon the cell type. The isolated RNA is analyzed by Northern gel andhybridization techniques with specific cDNA or PCR amplified probes.Northern analysis is quantified by densitometric scanning ofautoradiographs and normalization to housekeeping gene signals (G3PDH).Northern analysis may be supplemented with quantitative PCR analysisusing primers generated from the published cDNA sequences of the genesto be analyzed.

For Western blotting, solubilized protein lysates are isolated fromcells cultured on matrices containing osteogenic or chondrogenic agentsby standard techniques (Spiro R.C., et al., 1991, J. Cell. Biol.,115:1463-1473). After the lysis of cells the matrix is extracted instronger denaturants (8 M urea, GnHCL) to remove and examine bound orincorporated proteins. Protein samples are analyzed by standard Westernblotting techniques using specific polyclonal or monoclonal antibodies.

For metabolic cell labeling, cells cultured on a matrix aremetabolically radiolabeled with 35SO4, 35S-methionine or 3H/14C-labeledamino acids by standard techniques (Spiro et al., supra). Solubilizedcellular and matrix-associated proteins are quantitativelyimmunoprecipitated with antibodies specific for the protein of interestand analyzed by SDS-PAGE (Spiro et al., supra). Quantitation of resultsare performed by densitometric scanning of autoradiographs and signalswill be normalized to either cell equivalents or to a house-keepingprotein such as actin.

Additionally, the ability of a matrix of the present invention tosupport chondrogeneic differentiation in vivo may be tested in an inbredrat soft tissue implant model. Rat bone marrow or stromal cellsdescribed above are seeded onto the carrier at high density, culturedovernight in MEM medium containing 10% FBS serum and antibiotics, thentransferred into Millipore diffusion chambers and implantedintraperitoneally or subcutaneously into 8 week-old recipients. Chambersare harvested after 3 weeks and evaluated histologically for. cartilageformation.

A transplantation model in outbred rats is used to evaluate the abilityof the matrix to maintain the cartilage phenotype in vivo. Rib costalcartilage chondrocytes are seeded onto the carrier at high density andcultured overnight in Hams F-12 containing 1% rat serum and antibiotics.The seeded carriers are then implanted into posterior tibial musclepouches created by blunt dissection in 8 week-old male Sprague-Dawleyrats. Explants are taken at 14 and 28 days and evaluated histologicallyfor compatibility, cartilage growth, and maintenance of thedifferentiated phenotype based on staining for aggrecan and type IIcollagen.

For the in vivo tests, a matrix may be evaluated for the capabilitiesfor supporting osseous healing in a rat cranial defect model byimplantation into a 5 mm by 3 mm defect created in the parietal bone of6 weeks old male Sprague-Dawley rats. The defects are evaluated at 28days by radiographic and histologic analysis.

The in vivo model for cartilage repair is a full-thickness articularcartilage defect in the rabbit (Amiel et al., 1985, J. Bone Joint Surg.67A:911). Defects measuring approximately 3.7 mm in diameter and 5 mmdeep defect are created in the center of the medial femoral condyles ofadult male New Zealand white rabbits. The defects are then either filledwith the matrix or left unfilled as controls. The defects are evaluatedmorphologically and histologically at 6 and 12 weeks and then at 6months and one year.

The following examples are provided for purposes of illustration and arenot intended to limit the invention in any way.

EXAMPLE I

Preparation of a COL/HA bilayer matrix with 70% COL content in one layerand 100% HA content in another layer. This example illustrates how tocross-link a HA/DVS layer with a COL with HA gradient.

To 20 ml COL/HA suspension (560 mg of COL, Prep F fibers; 240 mg HA;0.2N NaOH) added with 240 mg of DVS. The mixture was immediately blendedusing a heavy duty blender at low speed for 2×5 sec., and poured to adesigned mold. After about 20 min. when the COL/HA slurry started togel, 10 ml of HA/DVS viscose containing 400 mg HA and DVS was added ontothe top of the COL/HA slurry. Since HA/DVS gels shortly after mixing,the viscose should be prepared only 4-5 min. before application by avigorous vortexing. The mold with its content was allowed to sit onbench at room temperature for one hour to gel completely, then placed in10% isopropyl alcohol solution for one hour. The matrix thus formed waswashed with a large volume of D.I. water with several changes for 48hours, followed by lyophilization.

EXAMPLE 2

Preparation of a COUHA bilayer matrix with 100% COL content in one layerand 100% HA content in another layer. This example illustrates how tocross-link a HA/DVS layer to a COL/Glutaraldehyde layer.

COL matrix was prepared by cross-linking pre-fabricated COL sponge withglutaraldyhyde in 30% isopropyl alcohol by a regular procedure adoptedin-house. The matrix was soaked in 0.2 N NaOH for 5 min. and placed inan appropriate mold. HA/DVS viscose was prepared as described in ExampleI and poured on the top of the COL matrix. After sitting on bench atroom temperature for one hour, the matrix was immersed in 10% isopropylalcohol for one hour, then large volumes of D.I. H₂O with severalchanges for 48 hours, followed by lyophilization.

EXAMPLE 3

Preparation of a COL/HA bilayer matrix with 100% COL content in onelayer and 100% HA content in another layer. This example illustrates howto cross-link a HA/DVS layer to a COL/DVS layer.

COL matrix was prepared by blending COL fiber (4%, 0.2 N NaOH) with DVSusing a heavy duty blender at the low speed for 2×5 sec. The COL/DVSslurry thus formed was poured into an appropriate mold and allowed tosit on bench at room temperature for 30 min. HA/DVS viscose was preparedas described in Example I and poured on the top of the COL/DVS gel.After sitting on bench at room temperature for an additional hour, thematrix was lyophilized. The dried matrix was immersed in 10% isopropylalcohol for one hour, then large volume of D.I. H₂O with several changesfor 48 hours, followed by lyophilization.

EXAMPLE 4

Preparation of a HA/DVS bilayer matrix with different cross-linkingdensity in its two layers. This example illustrates the method toprepare HA/DVS bilayer get with a higher porosity in one layer and alower one in the other through controlling their cross-linking degree.

400 mg HA in 10 ml 0.2 N NaOH was mixed with 400 μl DVS, mixedthoroughly, poured to a designed mold, then allowed to get at roomtemperature for 2 min. At this moment, 10 ml HA/DVS viscose (8%cross-linker and 4% HA) was poured onto the top of the HA gel previouslymolded. The bilayer gel continued to incubate at room temperature foranother hour to allow the gelation to be completed. The gel was washedand lyophilized as described above.

EXAMPLE 5

Preparation of COL/HA bilayer matrix with different mass density in itstwo layers. This example illustrates the method to prepare HA/DVSbilayer gel with a higher porosity in one layer and a lower one in theother through controlling their mass density.

A COL/HA slurry [d, 70 mg/ml; 9:1(COL/HA); 0.2 N NaOH] was mixed withDVS (75 mg/ml) by a vigorous blending, poured into a designed mold.Immediately, another part of COL/HA slurry with a lower mass density [d,35 mg/ml; 9:1(CO/HA); 0.2 N NaOH] was mixed with DVS (35 mg/ml) andpoured on the top of the first COL/HA gel. The matrix was then incubatedat room temperature for another hour followed by lyphilization. Thedried matrix was washed with a large volume of D.I. water andre-lyophilized.

EXAMPLE 6

Preparation of a COL/HA matrix with cross-linked HA in the core and 100%COL in the outside layer. This example illustrates how to embed a HAnetwork into a COL gel using acid COL solution.

A HA/DVS matrix with the diameter of 8 mm, which was pre-swelled in PBS,was placed in the center of a well of 12 well tissue culture plate. 1.60ml COL solution (Collagen Corporation, 3 mg/ml, 0.012 M HCl), 0.20 ml10× PBS, and 0.20 ml 0.10 N NaOH were added into a 4 ml polypropyleneround bottom tube, vortexed for 5 min., then incubated in a water bathat 37° C. The viscosity of the COL solution increased as time passed,and it finally became hard to flow along the wall when the tube wasplaced against both at a 60° angle from horizon. The viscose wascarefully poured into the well and the plate was incubated at 37° C. foran additional 40 min., COL gel formed filling the space between the coreHA matrix and the wall of the well. In this process, the penetration ofCOL viscose into the HA/DVS layer is dependent on its viscosity.Therefore, the COL viscose can be easily designed to only slightlypenetrate into the core and gel only at the edge to create a firmphysical cross-linking, while the integrity of two layers remains.

EXAMPLE 7

Preparation of COL/HA matrix with a COL gel in the core and HA/DVS inthe outside layer. This example illustrates how to get a COL gel layerinside a HA/DVS matrix using acid COL solution.

A HA/DVS matrix disc in the size of 8×4 mm (r×d) was punched with a hole(r, 4 mm) in the center, swelled in PBS and placed in a tissue cultureplate. A COL viscose prepared as described above was carefully pouredinto the hole. The plate continued incubating at 37° C. for anadditional 40 min.

EXAMPLE 8

Identification of COL/HA bilayer matrices.

Synthetic COL/HA bilayer matrices were identified for their stabilityand structural appearance.

For the stability study, matrices thus prepared were immersed in PBScontaining 1% penicillin/streptomycin and stored at 4° C. to preventenzymatic degradation. After 2 months, no dissociation of two layerscould be observed over all tested matrices, showing the stability ofbilayer matrices thus formed.

Matrices prepared as illustrated above were stained with 0.01% toluidineblue, then with eosin. Two colors, blue and purple, were shown in the HAdoman and COL domain respectively, indicating the formation of matrixwith two separate layers.

EXAMPLE 9

Culture of COL/HA bilayer matrix seeded with FRC cell. This exampleillustrates the effect of the COL/HA ratio in two separate layers oncell attachment and differentiation.

Fetal rat calavarial (FRC) cells were seeded on a COL/HA bilayer matrixprepared as described in Example 6 at the density of 1.5×10⁶ cell per mlHA/COL gel. 2.5 ml DMEM containing 2 μg GDF-5 was added to each well andcultured under traditional conditions. After 24 hours, the medium waspipeted out, the matrix was washed with PBS, fixed with 10% formaline,stained with crystal violet, then examined by microscope observation. Itwas found that the cells attached in the COL domain were spread andthose which attached in HA domain remained round.

EXAMPLE 10

Culture of COL/HA bilayer matrix pre-seeded with FRC cells. Thisillustrates the effect of differences in the composition of the twolayers on cell differentiation. Matrix prepared as described in example3 was used. One bilayer matrix was soaked in a solution of FGF (+GF),and a second bilayer matrix was implanted without use of FGF (−GF).Matrix was cut to cubes of the size 8×6×4 mm (L, W, H), sterilized withethanol, loaded with FRC cells at the density of 4×10⁵ cell/specimen,and cultured at 37° C. in DMEM for 4 weeks. The medium was changed everyother day. After 4 weeks, the matrix was removed from the medium, washedwith PBS, and examined for cellular morphology by histology, counted forcell number by quantitation of DNA, measured for levels of alkalinephosphatase activity (ALP) by reacting with p-nitrophenol, and measuredfor sulfated glycosaminoglycans (GAGs) by the dimethylmethlene blueassay. Cells in the HA layer had a round, aggregated, andchondrocyte-like morphology, while those grown in the COL layer wereflattened and spread. Biochemical analysis demonstrated that cells inthe COL layer expressed a high level of ALP and a low level of GAGscompared to those in the HA layer (Table 1). These results demonstratethat the differentiation of cells within distinct regions of the bilayermatrix can be determined by specific compositional changes.

Samples Cell proliferation* ALP** Sulfate GAG COL layer (+GF) 6.88 61.80.45 ± 0.03 COL layer (−GF) 6.42 103 −0.02 ± 0.06   HA layer (+GF) 5.537 2.53 ± 0.08 HA layer (−GF) 4.58 13 0.9 ± 0.1 *Cell proliferation, therate of DNA amount in each specimen at day 28 to that at day 1. **ALP,alkaline phosphatase activity, μmole/gDNA/min ***The O.D. at 595 nm of 5times diluted de-stained solution

1. A method for preparing a porous biodegradable multilayer matrix fortissue regeneration, said method comprising the step of applying a firstlayer comprising a porous polymeric component to a second layercomprising a porous polymeric component wherein said polymericcomponents are independently selected from the group consisting ofcovalently cross-linked proteins, covalently cross-linkedpolysaccharides, and proteins covalently cross-linked to polysaccharideswherein each of said layers has a porosity sufficient to accommodatelivings cells therein.
 2. The method of claim 1 wherein said first layercomprises two polysaccharides or proteins cross-linked to each other. 3.A method according to claim 1 wherein polymeric components areindependently selected from the group consisting of collagen, albumin,fibrinogen, fibronectin, vitronectin, laminin, hyaluronic acid, dextran,dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate,chitin, chitosan, heparan, heparan sulfate and alginate.
 4. The methodof claim 2 or 3 wherein said first layer comprises collagen cross-linkedto collagen.
 5. The method of claim 1 wherein said first layer comprisestwo different polysaccharides or proteins cross-linked to each other. 6.The method of claim 5 or 3 wherein said first layer compriseshyaluronate cross-linked to collagen.
 7. The method of claim 1 whereinsaid first polymeric layer is applied to said second layer by chemicalcross-linking with divinyl sulfone.
 8. A method according to claim 1further comprising the step of incorporating into said matrix at leastone growth factor, cDNA, gene construct, hormone or other biologicallyactive substance.
 9. A method according to claim 8 wherein said growthfactor, cDNA, gene construct, hormone, or other biologically activesubstance is incorporated after matrix fabrication.
 10. A methodaccording to claim 8 wherein said growth factor, cDNA, gene construct,hormone, or other biologically active substance is loaded during matrixfabrication.
 11. The method of claim 1 wherein said first layer isapplied to said second layer by thermal dehydration.